RESUMO
A diverse array of protocols have been established for the directed differentiation of human pluripotent stem cells (hPSCs) into a variety of cell types, including blood cells, for modeling development and disease, and for the development of cell-based therapeutics. These protocols recapitulate various signaling requirements essential for the establishment of the hematopoietic systems during embryonic development. However, in many instances, the functional properties of those progenitors, and their relevance to human development, remains unclear. The human embryo, much like other vertebrate model organisms, generates hematopoietic cells via successive anatomical location- and time-specific waves, each yielding cells with distinct functional and molecular characteristics. Each of these progenitor "waves" is characterized at the time of emergence of the direct hematopoietic progenitor in the vasculature, the hemogenic endothelial cell (HEC). Critically, despite decades of study in model organisms, the origins of each of these HEC populations remain unclear. Fortunately, through the directed differentiation of hPSCs, recent insights have been made into the earliest origins of each HEC population, revealing that each arises from transcriptionally and phenotypically distinct subsets of nascent mesoderm. Here, we outline the protocols to generate each mesodermal and HEC population via the formation of embryoid bodies and subsequent stage-specific signal manipulation. Through implementation of these discrete signal manipulations, it is possible to obtain human HEC populations that are exclusively extraembryonic-like or exclusively intraembryonic-like, enabling comparative developmental biology studies or specific translational applications.
RESUMO
Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.
Assuntos
Ceruletídeo/efeitos adversos , Proteínas Munc18/metabolismo , Pancreatite/metabolismo , Idoso , Animais , Ceruletídeo/metabolismo , Colecistocinina/efeitos adversos , Colecistocinina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Exocitose , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Munc18/genética , Pâncreas/metabolismo , Pancreatite/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMO
UNLABELLED: Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. SIGNIFICANCE: Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Glicosilases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores Etários , Animais , Linhagem Celular Tumoral , Análise por Conglomerados , Variações do Número de Cópias de DNA , Reparo do DNA , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Modelos Biológicos , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND & AIMS: During development of alcoholic pancreatitis, oxidative (acetaldehyde) and nonoxidative metabolites (ethyl palmitate, ethyl oleate), rather than ethanol itself, mediate toxic injury. Exposure of pancreatic acini to ethanol blocks cholecystokinin (CCK)-8-stimulated apical exocytosis and redirects exocytosis to the basolateral plasma membrane, causing interstitial pancreatitis. We examined how each ethanol metabolite contributes to these changes in exocytosis. METHODS: Rat pancreatic acini were incubated with concentrations of ethanol associated with alcoholic pancreatitis (20-50 mmol/L) or ethanol metabolites (1-3 mmol/L) and then stimulated with CCK-8. We performed single zymogen granule (ZG) exocytosis assays, Ca(2+) imaging studies, ultrastructural analyses (with electron microscopy), and confocal microscopy to assess the actin cytoskeleton and track the movement of vesicle-associated membrane protein (VAMP)-8-containing ZGs. Coimmunoprecipitation assays were used to identify complexes that contain the distinct combinations of Munc18 and the soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins, which mediate apical (ZG-apical plasma membrane) and basolateral exocytosis and fusion between ZGs (ZG-ZG). RESULTS: The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate reduced CCK-8-stimulated apical exocytosis and formation of apical exocytotic complexes (between Munc18b and Syntaxin-2, synaptosomal-associated protein of 23 kilodaltons [SNAP23], and VAMP2) in rat pancreatic acini. Acetaldehyde and ethyl oleate redirected CCK-8-stimulated exocytosis to the basal and lateral plasma membranes and translocation of VAMP8-containing ZGs toward the basolateral plasma membrane. This process was mediated primarily via formation of basolateral exocytotic complexes (between Munc18c and Syntaxin-4, SNAP23, and VAMP8). Exposure of the acini to acetaldehyde and ethyl oleate followed by CCK-8 stimulation mildly perturbed the actin cytoskeleton and Ca(2+) signaling; exposure to ethyl palmitate severely affected Ca(2+) signaling. Acetaldehyde, like ethanol, promoted fusion between ZGs by the formation of ZG-ZG exocytotic complexes (between Munc18b and Syntaxin-3, SNAP23, and VAMP8), whereas ethyl palmitate and ethyl oleate reduced ZG-ZG fusion and formation of these complexes. CONCLUSIONS: The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate perturb exocytosis processes in cultured rat pancreatic acini (apical blockade, basolateral exocytosis, and fusion between ZGs). Acetaldehyde and, to a lesser degree, ethyl oleate produce many of the same pathologic effects of ethanol on CCK-8-stimulated exocytosis in pancreatic acini.
Assuntos
Amilases/metabolismo , Etanol/toxicidade , Exocitose/efeitos dos fármacos , Pâncreas Exócrino/efeitos dos fármacos , Pancreatite Alcoólica/etiologia , Vesículas Secretórias/efeitos dos fármacos , Acetaldeído/metabolismo , Acetaldeído/toxicidade , Citoesqueleto de Actina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanol/metabolismo , Imunoprecipitação , Masculino , Fusão de Membrana/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Munc18/metabolismo , Ácidos Oleicos/metabolismo , Ácidos Oleicos/toxicidade , Ácidos Palmíticos/metabolismo , Ácidos Palmíticos/toxicidade , Pâncreas Exócrino/enzimologia , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/ultraestrutura , Pancreatite Alcoólica/enzimologia , Pancreatite Alcoólica/patologia , Proteínas Qa-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Secretórias/enzimologia , Vesículas Secretórias/metabolismo , Sincalida/farmacologia , Fatores de Tempo , Técnicas de Cultura de Tecidos , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.